The present invention relates to the production of frozen products, particularly foodstuffs and other biological products.
Hiterto, in order to achieve satisfactory freezing of foodstuffs and other biological products, freezing operations have had to be operated at temperatures well below those at which such food products would theoretically be expected to freeze in order to avoid problems of super-cooling. Such super-cooling can occur in both intra-and extra-cellular moisture present in foodstuffs and other biological products and leads to a failure of the product to freeze satisfactorily. While the degree of super-cooling may vary substantially from batch to batch, the risk that super-cooling may occur has led food freezing practitioners to operate plants at temperatures as low as -40.degree. C. in order to avoid such risks. Furthermore, in such operations, foods or other biological products to be frozen are typically kept at such low temperatures for excessive periods of time to ensure that problems resulting from super-cooling are avoided. Not only does the requirement to use very low temperatures for prolonged periods result in very high energy costs, but also a further problem that arises from super-cooling is a tendency for ice crystals in the frozen foodstuff to grow to a large size thereby diminishing the organoleptic and textural properties of the frozen food. Similarly, the thaw drip loss (normally regarded as a measure of the quality of the frozen product) may increase if excessive super-cooling occurs during freezing. Conventional practice has been to try to avoid ice crystal growth resulting from super-cooling by effecting quick freezing of products. Alternatively, use has been made of additives such as emulsifiers in ice cream or cryoprotectants such as glycerol or sugar in other products. These techniques, however, require cooling to very low temperatures with the consequence that such techniques have high energy requirements.
The effects of certain ice-nucleating active agents produced by various bacteria on the nucleation of ice crystals have recently attracted interest, primarily because of their potential for producing artificial snow for ski slopes and because of their role in frost damage of growing plants. In particular, the ice nucleating properties of Pseudomonas syringae have been widely reported. (See, for example, Maki, et al Applied Microbiology, September 1974, p. 456). In recent years, these properties have been investigated with particular emphasis on the role played by Pseudomonas syringae of a protein derived therefrom in causing frost damage to growing plants (see for example Lindow, Plant Disease, March 1983, p. 327). Considerable press attention has been given to the allowability of experiments in the open environment involving mutants of Pseudomonas syringae that have been mutated to eliminate the gene that is responsible for the production of ice-nucleating protein. It is hoped by allowing these mutants to compete with Pseudomonas syringae that are naturally present on plants that the population of ice nucleating Pseudomonas syringae may be reduced (see, for example, Lindow, et al Phytopathology Vol. 76, No. 10, p. 1069 Abstract 95 (1986).
Similar ice nucleating properties have been reported for several other microorganisms including Erwinia herbicola (see, for example, Lindow, et al Phytopathology 73 1097-1106 (1983)) and Kozloff, et al J. Bacteriology Jan. 1983 p. 222-231), Pseudomonas fluorescens (see, for example, Phelps, et al J. Bacteriology Aug. 1986 p. 496-502)) and Corotto, et al (EMBO Journal 5 231-236 (1986)) and Xanthomonas Camoestris (Derie and Schaail, Phytopathology 76 (10) p. 1117 (1986), Pseudomonas viridflava (Paulin, et al Proc 4th Int. Conf on Plant Pathogenic Bacteria Vol. 2 INRA Beaucoaze France 1978 Vol. 2 p. 725-731 and Anderson and Ashworth, Plant Physiol Vol. 80 pages 956-960 (1986)).
A number of investigations of the specific genes or proteins produced by them that are responsible for ice nucleation have been reported. For example, Green and Warner in a letter to Nature 317 p. 645 (1985) describe the determination of the sequence of the ice nucleation gene from Pseudomonas syringae which they called inaZ. They noted that this contains several repeats of a sequence reiteration with the consensus repeat having the sequence GCCGGTTATGGCAGCACGCTGACC, the gene having a total size of 4458. They also investigated whether deletion of fragments of inaZ affected the ability of the gene to produce an ice nucleating protein by inserting such modified genes into E Coli. It was found that in the case of deletions that did not result in a frameshift, ice nucleation properties were retained in a number of cases.
Corotti, et al in The EMBO Journal 5 p. 231-236 (1986) describe a DNA fragment of 75 kb obtained from Pseudomonas fluorescens that is capable of imparting ice L nucleating activity to E Coli. They designated their gene inaW. They also investigated the activity of inaW mutants and determined that insertions into a particular 3.9 kb sequence had particular effect on the activity of the gene. They concluded that the product of the gene, postulated to be a protein of about 180 kd molecular weight, was necessary to confer an ice nucleating (INA.sup.+) phenotype on E coli.
Kozloff, et al in J. Bacteriology 153 p. 222-231 (1983) hypothesize that the ice nucleating activity of Pseudomonas syringae and E herbicola stems from the presence of nucleating sites on their cell walls. Their results indicate that Pseudomonas syringae probably have 4-8 sites per cell and E herbicola 2 sites per cell.
Kozloff, et al in Science 226 845-846 (1984) suggest that a lipid phosphatidylinositol is present as well as a protein at ice nucleating sites or Pseudomonas syringae and Erwinia herbicola. Current thinking, however, now doubts this hypothesis.
Wobler, et al in Proc. Natl. Acad. Sci. USA 83 7256-7260 (1986) report the isolation of an ice nucleation protein from E. coli that have been transformed with a plasmid containing the inaZ gene obtained form Pseudomonas syringae. The protein (p 153) has an apparent molecular weight of 153 kd. The amino acid content of p153 corresponded closely with that predicted as the product of inaZ and the opening sequence of p153 (Met-Asn-Leu-Asp-Lys-Ala-Leu-Val-Leu-) corresponded exactly with the sequence that would be predicted to be coded by the inaZ gene. They further reported that a 180 protein obtained by a similar technique using the inaW gene obtained from Pseudomonas fluorescens apparently had similar sequences and structures to p153 obtained using inaZ. They suggest that the INA.sup.+ phenotype is conferred on both Pseudomonas syringae and Pseudomonas fluorescens by such proteins and that these proteins act as templates for ice nucleation even in the absence of the phospholipids Kozloff indicated to be necessary.
In Biophysical Journal 49 293a (1986) Wolber and Warner report that in the ice nucleating protein obtained from Pseudomonas syringae, the majority of the sequence consists of interlaced 8, 16 and 48 amino acid repeats. The secondary structure is apparently a .beta.-sheet structure for repeated sequences punctuated by 5-6 turns per 48 amino acid unit. It is suggested that the protein folds into a regular structure built up from the 48 amino acid repeat and that this structure presents hydrogen bonding side chains that mimic the ice lattice.
Phelps, et al in J. Bacteriology 167 p. 496 (1986) indicate the proteinaceous nature of the ice nucleating material obtained from Erwinia herbicola, reporting that cell free ice nucleating agents could be obtained from outer membrane residues and that such agents could induce nucleation of water at temperatures in the range -2 to -10.degree. C. Makino in Ann Phytopath Soc. Japan 48 452-457 (1982) reports that ice nucleation activity was found in Pseudomonas marginalis but to a lesser extent than in Pseudomonas syringae.
In addition to studies on ice nucleating bacteria as noted above, studies have also been made of ice nucleation having a biogenic origin in other organisms. Thus, Duman and Horwath in Ann. Rev. Physiol. 45 261-70 (1983) review the role of ice nucleation proteins in the hemolymph of certain beetles in imparting freeze-tolerance to the beetles by inhibiting super-cooling and ensuring freezing of extra-cellular fluid at fairly high temperatures thereby reducing the risk of intracellular freezing. Such proteins are reported to exist in Vespula (which have 3-6 ice nucleation proteins) and Dendroides (having 1-2 ice nucleation proteins). The nature of the ice nucleating protein produced by Vespula maculala was investigated by Duman, et al in J. Comparative Physiology B 154 79-83 (1984).
Ice nucleating agents have also been reported as being produced by Heterocapsia niei (a marine dinoflagellate) by Fall and Schnell in J. Marine Research Vol. 43 pages 257-265 (1985).
Each of the above cited articles is incorporated herein by reference.
The use of bacteria having an INA.sup.+ phenotype (Erwinia ananas) to cause texturization of certain proteinaceous foods has been suggested by Arai and Watenabe in Agricultural and Biological Chemistry 50, 169-175 (1986). They added cells to aqueous dispersions or hydrogels of proteins and polysaccharides to convert bulk water into directional ice crystals at temperatures between -5 and 0.degree. C. to form anisotropically textured products. Such products were produced using raw egg white, bovine blood, soybean curd, milk curd, aqueous dispersions or slurries of soybean protein isolate, hydrogel of agar, corn starch paste and hydrogels of glucomannan and calcium glucomannan. Such products are slowly cooled to -5.degree. C. in an air bath in the presence of the bacterial cells. The frozen products were then vacuum freeze dried at -30.degree. C. before setting by steaming to form flake-like textures. By slicing the products at right angles to the plane of the flakes, a textured product may be obtained.
When used herein the term ice nucleating phenotype (INA.sup.+) means an ability to induce nucleation of super-cooled water at a temperature above -20.degree. C. For example, P syringae PV pisi has a nucleation temperature of -2.9.degree. C. according to Makino (Ann Phytopath Soc. Japan Vol. 48 pages 452-457 (1982)).
The term "non-toxic microorganism" when used herein means microorganism having no adverse effect on humans when ingested in the amounts that are likely to result from the use of such microorganism to assist in ice nucleation.